Patients with cystic fibrosis (pwCF) are highly susceptible to chronic pulmonary infections due to mutations in the CFTR gene. From early childhood, pwCF experience repeated lung infections and often develop chronic bacterial and/or fungal colonization. Among the most clinically relevant pathogens, Pseudomonas aeruginosa and Aspergillus fumigatus frequently co-infect and are associated with worse outcomes, including excessive IL-1β-driven inflammation and accelerated lung function decline. Here we investigated the mechanisms underlying inflammasome overactivation during super-infection. We found that inflammasome hyperactivation occurred across macrophage populations, was independent of exogenous priming, and required live co-infection with both pathogens. P. aeruginosa and A. fumigatus cooperatively activated the NLRP3 inflammasome, and this response required both caspase-1 and caspase-8. Unexpectedly, gasdermin D was dispensable for IL-1β release. Bacterial flagellin, type IV pili and the type III secretion system, as well as the fungal polysaccharide galactosaminogalactan (GAG), were each required for overactivation. Mechanistically, P. aeruginosa activated the MyD88–TLR pathway, enhancing macrophage responses and promoting ITGAM (CD11b) expression. Under fungal super-infection, macrophages secreted complement component C3, which may bound fungal surface and engaged the complement receptor C3R (CD11b/CD18). Downstream SYK and ERK signaling amplified inflammasome activation and IL-1β release. Single-cell transcriptomic analysis of pwCF broncho-alveolar lavage and lung samples supported coordinated upregulation of complement and inflammasome pathways during bacterial-fungal infection. Together, these findings identify a complement–inflammasome signaling axis that drives pathological inflammation during bacterial-fungal co-infection in airways of pwCF and may represent a therapeutic target.

Psoriasis is a chronic inflammatory skin disorder characterized by aberrant keratinocyte proliferation and immune cell infiltration with upregulation of inflammatory cytokines. Here, we examined the contribution of HCAR2 encoding for the short-chain fatty acid receptor GPR109A. Human and mouse RNA sequencing public datasets reveal elevated HCAR2 gene expression in psoriatic as compared with healthy skin, both in keratinocytes and myeloid cells. Immunostaining and flow cytometry of imiquimod-induced psoriatic-like lesions in Hcar2-mRFP reporter mice showed increased GPR109A expression by keratinocytes and inflammatory cells. GPR109A-deficient mice demonstrated a more severe imiquimod-induced psoriasis-like response than wild-type mice, with exacerbated epidermal hyperplasia, dermal inflammatory cell infiltration, and increased inflammatory mediators myeloperoxidase, CXCL5, LCN2, interleukin (IL)-1β, IL-6, IL-23, and IL-17A. Conversely, topical administration of sodium butyrate reduced imiquimod-induced skin inflammation in wild-type mice, but not in GPR109A-deficient mice. Mechanistically, GPR109A agonist butyrate inhibits histone deacetylase 3, thus inhibiting IL-1β and the inflammatory IL-1β/IL-23/IL-17A axis in imiquimod-induced skin inflammation. Therefore, GPR109A may have a protective role in psoriasis pathogenesis, supporting a potential therapeutic benefit of sodium butyrate administration or other GPR109A agonists for treating psoriasis.

STING gain-of-function (GOF) mutations cause STING-Associated Vasculopathy with onset in Infancy (SAVI), a severe autoinflammatory disease. Mice carrying STING GOF V154M mutation develop profound T cell lymphopenia, partly due to impaired thymic development. To investigate the mechanisms of peripheral T cell dysfunctions, we analyzed transcriptomic and phenotypic profiles of splenic T cells from these mice. We found a terminally exhausted T cell phenotype, established early in life upon entry into the periphery, independent of type I interferons and intrinsic STING activation in T cells or stromal cells. Mechanistically, naive T cells in the lymphopenic periphery experienced heightened stimulation of the IL-7 receptor and TCR, including NFAT pathway, a key factor in T cell exhaustion. Transplantation of STING GOF hematopoietic stem cells with wild-type bone marrow prevented exhaustion in this non-lymphopenic context, placing lymphopenia as a key driver. T cell exhaustion was also observed in lymphopenic mice carrying Rag1 hypomorphic mutations. In conclusion, our results highlight T cell exhaustion induced by lymphopenia and could have important implications for the management of patients with severe immune deficiencies.

IntroductionCystic fibrosis is a genetic disorder associated with mutations in the Cftr gene, resulting in the production of thick and sticky mucus that can lead to various health complications, primarily affecting the respiratory and digestive systems. Pseudomonas aeruginosa and Aspergillus fumigatus are frequently identified microorganisms in cystic fibrosis (CF) patients. Superinfection by A. fumigatus in patients already colonized by P. aeruginosa causes hypersecretion of the inflammasome-dependent cytokine IL-1β. Unfortunately, high IL-1β release contributes to pulmonary damage in CF patients and decreases lung functions. The detrimental consequences of inflammasome overactivation pose a significant threat to CF patients. Therefore, more studies are needed to fully understand the complex interactions between inflammasomes, bacterial and fungal superinfections, and inflammation in CF.MethodsThe regulation of the inflammasome is studied in vitro during the superinfection by A. fumigatus of macrophages, derived from mice bone marrow and bronchoalveolar lavage, which are infected by the bacterium P. aeruginosa. The activation of the inflammasome is assessed through the analysis of caspase-1 protein cleavage, secretion of the cytokine IL-1β, and induction of cell death (pyroptosis) in infected and control macrophages. To decipher the molecular mechanisms enabling the overactivation of the inflammasome during a bacterial-fungal superinfection, wild-type and transgenic macrophages deleted in immune response and inflammasome signaling pathways, as well as inhibitors, have been used. We also explored the implication of the CFTR protein in inflammasome activation during the superinfection by using Cftr-/- and ΔF508d/d macrophages. Mutant bacterial and aspergillus strains are also employed to characterize the bacterial and fungal patterns involved in this inflammasome overactivation.ResultsIn our study, primary infection with P. aeruginosa showed a potentiation of macrophages allowing the overactivation of the inflammasome in response to a secondary infection with A. fumigatus. Indeed, fungal superinfection of these cells potentiated by the bacterium leads to greater NLRP3 inflammasome, caspases -1 and -8 activation, and heightened secretion of IL-1β. Results show that overactivation of the inflammasome during fungal superinfection is specific to a primary infection with the bacterium P. aeruginosa. The involvement of Cftr gene mutations in inflammasome activation during superinfection appears to depend on the mutation class. Different pathogen-associated molecular patterns (PAMPs) of P. aeruginosa and A. fumigatus are involved in this mechanism, such as type IV pili, flagellin, type II and type III secretion systems of P. aeruginosa, and galactosaminogalactan of A. fumigatus.ConclusionA primary infection with P. aeruginosa results in the potentiation of macrophages, enabling the overactivation of the inflammasome and an excessive secretion of IL-1β in response to a secondary infection by A. fumigatus. This could explain the worsening of pulmonary functions in co-infected patients. Results regarding a specific inflammasome response during superinfection based on Cftr mutations need to be confirmed. Overall, studying the immune response triggered by this interplay between bacteria and fungus in cystic fibrosis is crucial and further investigations are necessary.

Environmental air pollutants including ozone cause severe irritation and respiratory diseases. Here, we report that 6week's ozone exposure in mice (1.5ppm, twice weekly) causes airway hyperreactivity, eosinophil and neutrophil recruitment, Th2 immune response, respiratory barrier disruption with inflammation, fibrosis and emphysema reminiscent of COPD, more rapidly than cigarette smoke exposure. This model features important aspects of asthma-COPD overlap syndrome (ACOS) as recently described in patients. Since Tiotropium (TTP), an anticholinergic receptor antagonist, blocks smooth muscle cell contraction and mucus secretion with a prolonged bronchodilator effect in patients with asthma or COPD, we asked whether its effect is limited to bronchodilation. We report here that Tiotropium not only reduced airways hyperreactivity, but also drastically diminished eosinophil recruitment, Th2 cell response and ozone-induced lung inflammatory pathology including emphysema. Therefore, chronic O3-induced lung pathology in mice mimics ACOS in patients and is attenuated by TTP treatment. The mechanisms of TTP protective effect on respiratory barrier disruption and chronic inflammation need to be further explored.

Psoriasis is a chronic and recurrent inflammatory skin disease characterized by abnormal proliferation and differentiation of keratinocytes and activation of immune cells. However, the molecular driver that triggers this immune response in psoriatic skin remains unclear. The inflammation-related gene absent in melanoma 2 (AIM2) was identified as a susceptibility gene/locus associated with psoriasis. In this study, we investigated the role of AIM2 in the pathophysiology of psoriasis. We found elevated levels of mitochondrial DNA in patients with psoriasis, along with high expression of AIM2 in both the human psoriatic epidermis and a mouse model of psoriasis induced by topical imiquimod (IMQ) application. Genetic ablation of AIM2 reduced the development of IMQ-induced psoriasis by decreasing the production of type 3 cytokines (such as IL-17A and IL-23) and infiltration of immune cells into the inflammatory site. Furthermore, we demonstrate that IL-17A induced AIM2 expression in keratinocytes. Finally, the genetic absence of inflammasome components downstream AIM2, ASC, and caspase-1 alleviated IMQ-induced skin inflammation. Collectively, our data show that AIM2 is involved in developing psoriasis through its canonical activation.

IntroductionGround-level ozone is an important gaseous constituent of air pollution that contributes to lung disease progression and mortality. Ozone exposure in mice causes pulmonary inflammation evolving into lung emphysema and/or fibrotic patterns but the mechanisms are not well understood [1]. We investigated the role of the poorly characterized innate receptor nucleotide-binding domain and leucine-rich repeat containing protein 6 (NLRP6) involved in inflammasome scaffold [2], in ozone exposure-induced in the context of immunogenic cell death [3].MethodsUsing a chronic ozone exposure model of chronic obstructive pulmonary disease (COPD) in mice, we investigated the role of the NLRP6 receptor in pulmonary inflammation, emphysema and fibrosis by exposing wild-type, Nlrp6 deficient mice and mice deficient for Nlrp6 specifically in lung epithelial cells. In addition, we analyzed NLRP6 expression in lung using NLRP6 FLAG-tagged mice. NLRP6-dependent expression and/or activation of proteins characteristic of immunological cell deaths such as pyroptosis, apoptosis and necroptosis were analyzed by western blotting and immunofluorescence.ResultsWe observed that mouse chronic ozone exposure increased NLRP6 expression in bronchial and alveolar epithelial cells and in a lesser extend in airway macrophages. Interestingly Nlrp6 deficiency dampened pulmonary inflammation and alveolar damage with reduced neutrophil and eosinophil influxes, attenuated chemokine/cytokine and remodeling factor production, collagen deposition and lung fibrosis. Chronic ozone-induced a loss of alveolar type 1 pneumocytes that was attenuated in Nlrp6 deficient mice. Mechanistically, we report that chronic ozone exposure promoted NLRP6-dependent caspase-1, caspase-11 and gasdermin D activation in alveolar type 1 pneumocytes. Chronic ozone exposure also induced NLRP6-dependent expression of apoptotic and necroptotic markers in lung tissue.ConclusionWe identified NLRP6 as a new innate sensor of chronic ozone-induced lung injury promoting pulmonary inflammation leading to emphysema and fibrosis in mice. Our results suggest that chronic ozone induces pulmonary inflammation through NLRP6 inflammasome-dependent pyroptosis and necroptosis of alveolar type 1 pneumocytes. Understanding the mechanisms of pollutant-induced alveolar cell death, lung inflammation and repair might help fight COPD and lung fibrosis.

Chronic obstructive pulmonary disease (COPD) is a major health issue primarily caused by cigarette smoke (CS) and characterized by breathlessness and repeated airway inflammation. NLRP6 is a cytosolic innate receptor controlling intestinal inflammation and orchestrating the colonic host–microbial interface. However, its roles in the lungs remain largely unexplored. Using CS exposure models, our data show that airway inflammation is strongly impaired in Nlrp6-deficient mice with drastically fewer recruited neutrophils, a key cell subset in inflammation and COPD. We found that NLRP6 expression in lung epithelial cells is important to control airway and lung tissue inflammation in an inflammasome-dependent manner. Since gut-derived metabolites regulate NLRP6 inflammasome activation in intestinal epithelial cells, we investigated the link between NLRP6, CS-driven lung inflammation, and gut microbiota composition. We report that acute CS exposure alters gut microbiota in both wild-type (WT) and Nlrp6-deficient mice and that antibiotic treatment decreases CS-induced lung inflammation. In addition, gut microbiota transfer from dysbiotic Nlrp6-deficient mice to WT mice decreased airway lung inflammation in WT mice, highlighting an NLRP6-dependent gut-to-lung axis controlling pulmonary inflammation.

Introduction:The pathogenesis of chronic lung diseases is multifaceted with a major role of recurrent micro-injuries of the epithelium. While several reports clearly indicated a prominent role for surfactant-producing alveolar epithelial type 2 (AT2) cells, the contribution of gas exchange-permissive alveolar epithelial type 1 (AT1) cells has not been addressed yet. Here, we investigated whether repeated injury of AT1 cells leads to inflammation and interstitial fibrosis.Methods:We chose an inducible model of AT1 cell depletion following local diphtheria toxin (DT) administration using an iDTR flox/flox (idTRfl/fl) X Aquaporin 5CRE (Aqp5CRE) transgenic mouse strain.Results:We investigated repeated doses and intervals of DT to induce cell death of AT1 cells causing inflammation and interstitial fibrosis. We found that repeated DT administrations at 1ng in iDTRfl/fl X Aqp5CRE mice cause AT1 cell death leading to inflammation, increased tissue repair markers and interstitial pulmonary fibrosis.Discussion:Together, we demonstrate that depletion of AT1 cells using repeated injury represents a novel approach to investigate chronic lung inflammatory diseases and to identify new therapeutic targets.

Chronic obstructive pulmonary disease (COPD) is a major health issue not only due to cigarette smoking but also to air pollution, in particular ozone exposure that is expected to increase in the context of global warming. COPD is characterized by progressive airway obstruction and impaired immune response to bacterial and viral infections. Pollutants induce pulmonary damage, chronic inflammation, and mucus hypersecretion leading to bronchial obstruction, alveolar wall destruction, and strongly impairing lung functions. The underlying mechanisms resulting in chronic inflammation, exacerbation, and impaired lung function remain poorly understood. However, oxidative stress and host-derived danger signals are common triggers leading to cigarette smoke (CS)- or ozone-induced lung injury and inflammation notably by activating inflammasome pathways. Inflammasomes are cytoplasmic multiprotein complexes involved in the regulation of homeostasis and inflammation. Their activation leads to maturation and secretion of major proinflammatory cytokines IL-1β and IL-18 as well as gasdermin D-mediated pore formation. Here we review recent studies analyzing the role of the inflammasomes in human and animal models of CS- or ozone-exposure in the development of pulmonary inflammation potentially leading to COPD and/or fibrosis.

Chronic pulmonary inflammation and chronic obstructive pulmonary disease (COPD) are major health issues largely due to air pollution and cigarette smoke (CS) exposure. The role of the innate receptor NLRP3 (nucleotide-binding domain and leucine-rich repeat containing protein 3) orchestrating inflammation through formation of an inflammasome complex in CS-induced inflammation or COPD remains controversial. Using acute and subchronic CS exposure models, we found that Nlrp3 -deficient mice or wild-type mice treated with the NLRP3 inhibitor MCC950 presented an important reduction of inflammatory cells recruited into the bronchoalveolar space and of pulmonary inflammation with decreased chemokines and cytokines production, in particular IL-1β demonstrating the key role of NLRP3. Furthermore, mice deficient for Caspase-1 / Caspase-11 presented also decreased inflammation parameters, suggesting a role for the NLRP3 inflammasome. Importantly we showed that acute CS-exposure promotes NLRP3-dependent cleavage of gasdermin D in macrophages present in the bronchoalveolar space and in bronchial airway epithelial cells. Finally, Gsdmd -deficiency reduced acute CS-induced lung and bronchoalveolar space inflammation and IL-1β secretion. Thus, we demonstrated in our model that NLRP3 and gasdermin D are key players in CS-induced pulmonary inflammation and IL-1β release potentially through gasdermin D forming-pore and/or pyroptoctic cell death.

The cGAS–STING pathway displays important functions in the regulation of innate and adaptive immunity following the detection of microbial and host-derived DNA. Here, we briefly summarize biological functions of STING and review recent literature highlighting its important contribution in the context of respiratory diseases. Over the last years, tremendous progress has been made in our understanding of STING activation, which has favored the development of STING agonists or antagonists with potential therapeutic benefits. Antagonists might alleviate STING-associated chronic inflammation and autoimmunity. Furthermore, pharmacological activation of STING displays strong antiviral properties, as recently shown in the context of SARS-CoV-2 infection. STING agonists also elicit potent stimulatory activities when used as an adjuvant promoting antitumor responses and vaccines efficacy.

Idiopathic pulmonary fibrosis (IPF) is the most common and severe type of interstitial lung disease for which current treatments display limited efficacy. IPF is largely driven by host-derived danger signals released upon recurrent local tissue damage. Here we explored the roles of self-DNA and stimulator of interferon genes (STING), a protein belonging to an intracellular DNA sensing pathway that leads to type I and/or type III interferon (IFN) production upon activation. Using a mouse model of IPF, we report that STING deficiency leads to exacerbated pulmonary fibrosis with increased collagen deposition in the lungs and excessive remodeling factors expression. We further show that STING-mediated protection does not rely on type I IFN signaling nor on IL-17A or TGF-β modulation but is associated with dysregulated neutrophils. Together, our data support an unprecedented immunoregulatory function of STING in lung fibrosis.

Innate immunity is regulated by a broad set of evolutionary conserved receptors to finely probe the local environment and maintain host integrity. Besides pathogen recognition through conserved motifs, several of these receptors also sense aberrant or misplaced self-molecules as a sign of perturbed homeostasis. Among them, self-nucleic acid sensing by the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway alerts on the presence of both exogenous and endogenous DNA in the cytoplasm. We review recent literature demonstrating that self-nucleic acid detection through the STING pathway is central to numerous processes, from cell physiology to sterile injury, auto-immunity and cancer. We address the role of STING in autoimmune diseases linked to dysfunctional DNAse or related to mutations in DNA sensing pathways. We expose the role of the cGAS/STING pathway in inflammatory diseases, neurodegenerative conditions and cancer. Connections between STING in various cell processes including autophagy and cell death are developed. Finally, we review proposed mechanisms to explain the sources of cytoplasmic DNA.

Cigarette smoke (CS) is the major cause of chronic lung injuries, such as chronic obstructive pulmonary disease (COPD). In patients with severe COPD, tertiary lymphoid follicles containing B lymphocytes and B cell-activating factor (BAFF) overexpression are associated with disease severity. In addition, BAFF promotes adaptive immunity in smokers and mice chronically exposed to CS. However, the role of BAFF in the early phase of innate immunity has never been investigated. We acutely exposed C57BL/6J mice to CS and show early BAFF expression in the bronchoalveolar space and lung tissue that correlates to airway neutrophil and macrophage influx. Immunostaining analysis revealed that neutrophils are the major source of BAFF. We confirmedin vitrothat neutrophils secrete BAFF in response to cigarette smoke extract (CSE) stimulation. Antibody-mediated neutrophil depletion significantly dampens lung inflammation to CS exposure but only partially decreases BAFF expression in lung tissue and bronchoalveolar space suggesting additional sources of BAFF. Importantly, BAFF deficient mice displayed decreased airway neutrophil recruiting chemokines and neutrophil influx while the addition of exogenous BAFF significantly enhanced this CS-induced neutrophilic inflammation. This demonstrates that BAFF is a key proinflammatory cytokine and that innate immune cells in particular neutrophils, are an unconsidered source of BAFF in early stages of CS-induced innate immunity.

The study of Huppertz and co-workers addresses the role of the NLRP3 inflammasome in sarcoidosis. Employing both mouse model and human samples, they provide evidence of NLPR3 inflammasome activation and increased IL-1β production in lung granulomas.http://bit.ly/32a4GsI

Cigarette smoke exposure is a leading cause of chronic obstructive pulmonary disease (COPD), a major health issue characterized by airway inflammation with fibrosis and emphysema. Here we demonstrate that acute exposure to cigarette smoke causes respiratory barrier damage with the release of self-dsDNA in mice. This triggers the DNA sensor cGAS (cyclic GMP-AMP synthase) and stimulator of interferon genes (STING), driving type I interferon (IFN I) dependent lung inflammation, which are attenuated in cGAS, STING or type I interferon receptor (IFNAR) deficient mice. Therefore, we demonstrate a critical role of self-dsDNA release and of the cGAS-STING-type I interferon pathway upon cigarette smoke-induced damage, which may lead to therapeutic targets in COPD.

Necrotic cell death during Mycobacterium tuberculosis (Mtb) infection is considered host detrimental since it facilitates mycobacterial spread. Ferroptosis is a type of regulated necrosis induced by accumulation of free iron and toxic lipid peroxides. We observed that Mtb-induced macrophage necrosis is associated with reduced levels of glutathione and glutathione peroxidase-4 (Gpx4), along with increased free iron, mitochondrial superoxide, and lipid peroxidation, all of which are important hallmarks of ferroptosis. Moreover, necrotic cell death in Mtb-infected macrophage cultures was suppressed by ferrostatin-1 (Fer-1), a well-characterized ferroptosis inhibitor, as well as by iron chelation. Additional experiments in vivo revealed that pulmonary necrosis in acutely infected mice is associated with reduced Gpx4 expression as well as increased lipid peroxidation and is likewise suppressed by Fer-1 treatment. Importantly, Fer-1-treated infected animals also exhibited marked reductions in bacterial load. Together, these findings implicate ferroptosis as a major mechanism of necrosis in Mtb infection and as a target for host-directed therapy of tuberculosis.

Idiopathic pulmonary fibrosis is a progressive, devastating, and yet untreatable fibrotic disease of unknown origin. Interleukin-33 (IL-33), an IL-1 family member acts as an alarmin with pro-inflammatory properties when released after stress or cell death. Here, we investigated the role of IL-33 in the bleomycin (BLM)-induced inflammation and fibrosis model using mice IL-33 receptor [chain suppression of tumorigenicity 2 (ST2)] mice compared with C57BL/6 wild-type mice. Unexpectedly, 24 h post-BLM treatment ST2-deficient mice displayed augmented inflammatory cell recruitment, in particular by neutrophils, together with enhanced levels of chemokines and remodeling factors in the bronchoalveolar space and/or the lungs. At 11 days, lung remodeling and fibrosis were decreased with reduced M2 macrophages in the lung associated with M2-like cytokine profile in ST2-deficient mice, while lung cellular inflammation was decreased but with fluid retention (edema) increased. In vivo magnetic resonance imaging (MRI) analysis demonstrates a rapid development of edema detectable at day 7, which was increased in the absence of ST2. Our results demonstrate that acute neutrophilic pulmonary inflammation leads to the development of an IL-33/ST2-dependent lung fibrosis associated with the production of M2-like polarization. In addition, non-invasive MRI revealed enhanced inflammation with lung edema during the development of pulmonary inflammation and fibrosis in absence of ST2.